Cortistatin polypeptides

ABSTRACT

The present invention relates to novel cortistatin polypeptides, and to polynucleotides encoding these polypeptides. More specifically, isolated nucleic acid molecules are provided encoding a Human Cortistatin polypeptide. Cortistatin polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting variations in Human Cortistatin gene expression and therapeutic methods for treating individuals in need of an increased level of Human Cortistatin activity.

[0001] This application claims the benefit of the filing date ofprovisional applications 60/033,980, filed on Dec. 31, 1996, and60/037,386, filed on Feb. 7, 1997, which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to novel cortistatin polypeptides,and to polynucleotides encoding these polypeptides. More specifically,isolated nucleic acid molecules are provided encoding a HumanCortistatin polypeptide. Cortistatin polypeptides are also provided, asare vectors, host cells and recombinant methods for producing the same.Also provided are diagnostic methods for detecting variations in HumanCortistatin gene expression and therapeutic methods for treatingindividuals in need of an increased level of Human Cortistatin activity.

[0004] 2. Related Art

[0005] The 1970's saw the initiation of a great amount of scientificdiscovery and research into the regulation of hormone release,especially by neuropeptides. Somatostatin, a 14 mer cyclic polypeptidewas one of these neuropeptide hormone regulators. It was discovered tobe released primarily in the hypothalamus, and to inhibit Growth Hormone(GH) secretion from the pituitary gland, and to decrease the GH responseto secretagogues. Somatostatin is also found throughout the brain andserves as a neurotransmitter in many areas, including the spinal cord,brain stem, and cerebral cortex. It is also present in thegastrointestinal tract, where the D cells of the pancreatic isletsregulate insulin and glucagon secretion.

[0006] There has been a lot of interest in studying the actions ofsomatostatin and analogues for efficacy in the therapy of acromegaly,secretory pancreatic tumors, pancreatitis, acute gastric ulcers andstress gastritis.

[0007] Thus, there is a continuing need in the art for isolating novelhormone-regulating peptides, especially neuropeptides of human origin,useful for therapy in various hormone mediated processes.

SUMMARY OF THE INVENTION

[0008] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the Human Cortistatin polypeptidehaving the amino acid sequence shown in FIG. 1 (amino acid residues −19to 86 in SEQ ID NO:2) or the amino acid sequence encoded by the cDNAclone deposited in a bacterial host as ATCC Deposit Number 97639 on Jun.27, 1996. The nucleotide sequence determined by sequencing the depositedCortistatin clone, which is shown in SEQ ID NO:1, contains an openreading frame encoding a polypeptide of 105 amino acid residues,including an initiation codon at positions 46-48 in SEQ ID NO:1, with aleader sequence of about 19 amino acid residues, and a predictedmolecular weight of about 12 kDa. The amino acid sequence of the matureCortistatin protein is shown in FIG. 1 (amino acid residues from about 1to about 86 in SEQ ID NO:2).

[0009] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the Human Cortistatin polypeptide having the complete aminoacid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding theHuman Cortistatin polypeptide having the amino acid sequence atpositions from about −18 to about 86 in SEQ ID NO:2; (c) a nucleotidesequence encoding the mature Human Cortistatin polypeptide having theamino acid sequence at positions from about 1 to about 86 in SEQ IDNO:2; (d) a nucleotide sequence encoding the polypeptide having theamino acid sequence at positions from about 58 to about 86 in SEQ IDNO:2; (e) a nucleotide sequence encoding the Human Cortistatinpolypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97639; (f) a nucleotide sequenceencoding the mature Human Cortistatin polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97639;and (g) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), or (f) above. Preferably, thenucleic acid molecule will encode the mature polypeptide in SEQ ID NO:2or encoded by the above-described deposited cDNA.

[0010] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 95% identical, and more preferably at least 96%, 97%,98% or 99% identical, to any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), or (g), above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide in (a),(b), (c), (d), (e), (f), or (g), above. This polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.

[0011] An additional nucleic acid embodiment of the invention relates toan isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of a HumanCortistatin polypeptide having an amino acid sequence in (a), (b), (c),(d), (e), (f), or (g), above.

[0012] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of Cortistatin polypeptides or peptides by recombinanttechniques.

[0013] The invention further provides an isolated Human Cortistatinpolypeptide having an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of the Human Cortistatinpolypeptide having the complete 105 amino acid sequence, including theleader sequence shown in SEQ ID NO:2; (b) the amino acid sequence of theHuman Cortistatin polypeptide lacking the N-terminal methionine residuehaving the amino acid sequence at positions from about −18 to about 86in SEQ ID NO:2; (c) the amino acid sequence of the mature HumanCortistatin polypeptide (without the leader) having the amino acidsequence at positions from about 1 to about 86 SEQ ID NO:2; (d) theamino acid sequence of the polypeptide having the amino acid sequence atpositions from about 58 to about 86 SEQ ID NO:2; (e) the amino acidsequence of the Human Cortistatin polypeptide having the complete aminoacid sequence, including the leader, encoded by the cDNA clone containedin ATCC Deposit No. 97639; and (f) the amino acid sequence of the matureCortistatin polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97639.

[0014] An additional embodiment of this aspect of the invention relatesto a peptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a Cortistatin polypeptide having an aminoacid sequence described in (a), (b), (c), (d), (e), or (f), above.Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a Cortistatin polypeptide of the inventioninclude portions of such polypeptides with at least six or seven,preferably at least nine, and more preferably at least about 30 aminoacids to about 50 amino acids, although epitope-bearing polypeptides ofany length up to and including the entire amino acid sequence of apolypeptide of the invention described above also are included in theinvention. In another embodiment, the invention provides an isolatedantibody that binds specifically to a Cortistatin polypeptide having anamino acid sequence described in (a), (b), (c), (d), (e), or (f) above.

[0015] The invention further provides methods for isolating antibodiesthat bind specifically to a Cortistatin polypeptide having an amino acidsequence as described herein.

[0016] The present inventors have discovered that Cortistatin isexpressed primarily in the brain, especially in the putamen. For anumber of disorders, significantly higher or lower levels of Cortistatingene expression can be detected in brain, especially putamen, tissue orbodily fluids (e.g., serum, plasma, urine, synovial fluid or spinalfluid) taken from an individual having such a disorder, relative to a“standard” Cortistatin gene expression level, i.e., the Cortistatinexpression level in brain tissue or bodily fluids from an individual nothaving the disorder. Thus, the invention provides a diagnostic methoduseful during diagnosis of a neurological disorder, which involves (a)assaying Cortistatin gene expression level in cells or body fluid ofthat individual; (b) comparing that Cortistatin gene expression levelwith a standard Cortistatin gene expression level, whereby an increaseor decrease in the assayed Cortistatin gene expression level compared tothe standard expression level is indicative of said neurologicaldisorder.

[0017] An additional aspect of the invention is related to a method fortreatment of an individual in need of an increased level of Cortistatinactivity in the body comprising administering to such an individual acomposition comprising an isolated Cortistatin polypeptide of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid(SEQ ID NO:2) sequences of the complete Human Cortistatin proteindetermined by sequencing of the HEBCI67X DNA clone contained in ATCCDeposit No. 97639. The protein has a leader sequence of about 19 aminoacid residues (underlined) and a deduced molecular weight of about 12kDa. The amino acid sequence of the predicted mature Human Cortistatinprotein is shown in FIG. 1 (last 86 amino acids) and in SEQ ID NO:2(amino acid residues from about 1 to about 86).

[0019] The nucleotide at position 314 in FIG. 1 (SEQ ID NO:1) may beeither an A or a G resulting in the encoded amino acid being either K(lysine) or R (arginine). Similarly, the nucleotide at position 342 inFIG. 1 (SEQ ID NO:1) may be an A or a G (conserved change), and thenucleotide at position 348 in FIG. 1 (SEQ ID NO:1) may be a T or a C(conserved change).

[0020]FIG. 2 shows the regions of similarity between the amino acidsequences of the Human Cortistatin protein (lower line) and the ratCortistatin (upper line) (SEQ ID NO:3). The sequence of the ratcortistatin is from de Lecea et al., Nature 381:242 (May 16, 1996).

[0021]FIG. 3 shows an analysis of the Human Cortistatin amino acidsequence. Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown. In the “Antigenic Index—Jameson-Wolf”graph, amino acid residues 23 to 38, 40 to 47, 58 to 65, 66 to 98 and100 to 105 in FIG. 1 (amino acid residues 4 to 19, 21 to 28, 39 to 46,47 to 79 and 81 to 86 in SEQ ID NO:2) correspond to the shown highlyantigenic regions of the Human Cortistatin protein.

[0022]FIG. 4 (SEQ ID NO:4) shows the sequence of a 29 amino acidsynthetic carboxy terminal peptide (upper line) spanning active fragmentof mature Human Cortistatin (lower line). The sequence of this 29 aminoacid fragment corresponds to amino acids 58 to 86 in SEQ ID NO:2.

DETAILED DESCRIPTION

[0023] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the Human Cortistatin proteinhaving the amino acid sequence shown in SEQ ID NO:2, which wasdetermined by sequencing a cloned cDNA. Human Cortistatin is a novelmember of the brain neuropeptides. The Human Cortistatin protein of thepresent invention shares sequence homology with the rat cortistatin(FIG. 2) (SEQ ID NO:3), which in turn has sequence homology withsomatostatin. The nucleotide sequence shown in SEQ ID NO:1 was obtainedby sequencing the HEBCI67X clone, which was deposited on Jun. 27, 1996at the American Type Culture Collection, 12301 Park Lawn Drive,Rockville, Md. 20852, and given accession number 97639. The depositedclone is contained in the pBluescript SK(−) plasmid (Stratagene,LaJolla, Calif.).

[0024] Nucleic Acid Molecules

[0025] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 95% identical,more typically at least about 96% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0026] Unless otherwise indicated, each “nucleotide sequence” set forthherein is presented as a sequence of deoxyribonucleotides (abbreviatedA, G, C and T). However, by “nucleotide sequence” of a nucleic acidmolecule or polynucleotide is intended, for a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, and for an RNAmolecule or polynucleotide, the corresponding sequence ofribonucleotides (A, G, C and U), where each thymidinedeoxyribonucleotide (T) in the specified deoxyribonucleotide sequence isreplaced by the ribonucleotide uridine (U). For instance, reference toan RNA molecule having the sequence of SEQ ID NO:1 set forth usingdeoxyribonucleotide abbreviations is intended to indicate an RNAmolecule having a sequence in which each deoxyribonucleotide A, G or Cof SEQ ID NO:1 has been replaced by the corresponding ribonucleotide A,G or C, and each deoxyribonucleotide T has been replaced by aribonucleotide U.

[0027] Using the information provided herein, such as the nucleotidesequence in FIG. 1 (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding a Cortistatin polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in SEQ ID NO:1 was discovered in acDNA library derived from human cerebellum. The determined nucleotidesequence of the Human Cortistatin cDNA of SEQ ID NO:1 contains an openreading frame encoding a protein of 105 amino acid residues with aninitiation codon at positions 46-48 of the nucleotide sequence shown inSEQ ID NO:1, and a predicted leader sequence of about 19 amino acidresidues, and a deduced molecular weight of about 12 kDa. The amino acidsequence of the predicted mature Human Cortistatin protein is shown inSEQ ID NO:2 amino acid residues from about 1 to about 86. The HumanCortistatin protein shown in SEQ ID NO:2 is about 56% identical andabout 90% similar to rat Cortistatin (FIG. 2). As one of ordinary skillwould appreciate, due to the possibilities of sequencing errorsdiscussed above, as well as the variability of cleavage sites forleaders in different known proteins, the actual Human Cortistatinpolypeptide encoded by the deposited cDNA comprises about 105 aminoacids, but may be anywhere in the range of 100-110 amino acids; and theactual leader sequence of this protein is about 19 amino acids, but maybe anywhere in the range of about 15 to about 25 amino acids.

[0028] As indicated, the present invention also provides the matureform(s) of the Human Cortistatin protein of the present invention.According to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the,rough endoplasmic reticulum has been initiated. Most mammalian cells andeven insect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species on the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature Human Cortistatin polypeptideshaving the amino acid sequence encoded by the cDNA clone contained inthe host identified as ATCC Deposit No. 97639 and as shown in SEQ IDNO:2. By the mature Human Cortistatin protein having the amino acidsequence encoded by the cDNA clone contained in the host identified asATCC Deposit No. 97639 is meant the mature form(s) of the HumanCortistatin protein produced by expression in a mammalian cell (e.g.,COS cells, as described below) of the complete open reading frameencoded by the human DNA sequence of the clone contained in the vectorin the deposited host. As indicated below, the mature Human Cortistatinhaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 97639 may or may not differ from the predicted “mature”Human Cortistatin protein shown in SEQ ID NO:2 (amino acid residues fromabout 1 to about 86) depending on the accuracy of the predicted cleavagesite based on computer analysis.

[0029] Methods for predicting whether a protein has a secretory leaderas well as the cleavage point for that leader sequence are availablebecause it is known that much of the cleavage specificity for asecretory protein resides in certain amino acid residues within thesignal sequence and the N-terminus of the mature protein, particularlyresidues immediately surrounding the cleavage site. For instance, themethod of McGeoch (Virus Res. 3:271-286 (1985)) uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje (NucleicAcids Res. 14:4683-4690 (1986)) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2 where +1indicates the amino acid terminus of the mature protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. von Heinje, supra.However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

[0030] In the present case, the predicted amino acid sequence of thecomplete Cortistatin polypeptides of the present invention were analyzedby a computer program (“PSORT”). This program is available from Dr.Kenta Nakai of the Institute for Chemical Research, Kyoto University(see K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is anexpert system for predicting the cellular location of a protein based onthe amino acid sequence. As part of this computational prediction oflocalization, the methods of McGeoch and von Heinje are incorporated.The analysis by the PSORT program predicted the cleavage sites betweenamino acids −1 and 1 in SEQ ID NO:2. Thereafter, the complete amino acidsequences were further analyzed by visual inspection, applying a simpleform of the (−1,−3) rule of von Heine. von Heinje, supra. Thus, theleader sequence for the Cortistatin protein is predicted to consist ofamino acid residues from about −19 to about −1 in SEQ ID NO:2, while thepredicted mature Cortistatin protein consists of residues from about 1to about 86.

[0031] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors, as well as the variability ofcleavage sites for leaders in different known proteins, the actualCortistatin polypeptide encoded by the deposited cDNA comprises about105 amino acids, but may be anywhere in the range of 100 to 110 aminoacids; and the actual leader sequence of this protein is about 19 aminoacids, but may be anywhere in the range of about 15 to about 25 aminoacids.

[0032] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0033] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0034] In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to an extensive portion of SEQ ID NO:1.These cDNA clones are designated HEBCI67R (SEQ ID NO:9) and HSVCB08RA(SEQ ID NO:10).

[0035] The sequence of a public EST, having GenBank Accession No.W79063, related to a portion of SEQ ID NO:1 is shown in SEQ ID NO:11.This public EST is 456 nucleotides in length and contains a region of373 nucleotides having a complementary sequence identical to nucleotides1 to 373 of the sequence shown in SEQ ID NO:1 with the exception ofseven mismatched nucleotides at positions 128, 161, 173, 306, 343, 345,and 400 in SEQ ID NO:11 and two undisclosed nucleotides at positions 163and 412 in SEQ ID NO:11. The undisclosed nucleotides in SEQ ID NO:11 arerepresented by the letter “N”.

[0036] In another aspect, the invention provides isolated nucleic acidmolecules encoding the Human Cortistatin polypeptide having an aminoacid sequence as encoded by the cDNA clone contained in the plasmiddeposited as ATCC Deposit No. 97639 on Jun. 27, 1996. In a furtherembodiment, nucleic acid molecules are provided encoding the matureHuman Cortistatin polypeptide or the full-length Human Cortistatinpolypeptide lacking the N-terminal methionine. The invention alsoprovides an isolated nucleic acid molecule having the nucleotidesequence shown in SEQ ID NO:1 or the nucleotide sequence of the HumanCortistatin cDNA contained in the above-described deposited clone, or anucleic acid molecule having a sequence complementary to one of theabove sequences. Such isolated molecules, particularly DNA molecules,are useful as probes for gene mapping, by in situ hybridization withchromosomes, and for detecting expression of the Human Cortistatin genein human tissue, for instance, by Northern blot analysis. As describedin detail below, detecting altered Human Cortistatin gene expression incertain tissues or bodily fluids is indicative of disorder(s) involvingthe hippocampus and cerebral cortex regions of the brain.

[0037] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in SEQ ID NO:1 isintended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course larger DNAfragments 50, 100, 150, 200, 250, 300, 350, 400, or 425 nt in length ofthe sequence shown in SEQ ID NO:1 are also useful according to thepresent invention as are fragments corresponding to most, if not all, ofthe nucleotide sequence of the cDNA clone contained in the plasmiddeposited as ATCC Deposit No. 97639 or as shown in SEQ ID NO:1. By afragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in SEQ IDNO:1. Since the gene has been deposited and the nucleotide sequenceshown in SEQ ID NO:1 is provided, generating such DNA fragments would beroutine to the skilled artisan. For example, restriction endonucleasecleavage or shearing by sonication could easily be used to generatefragments of various sizes. Alternatively, such fragments could begenerated synthetically.

[0038] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the HumanCortistatin protein. In particular, such nucleic acid fragments of thepresent invention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 4 to about 19 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 21 to about 28in SEQ ID NO:2; a polypeptide comprising amino acid residues from about39 to about 46 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 47 to about 79 in SEQ ID NO:2; and a polypeptidecomprising amino acid residues from about 81 to about 86 in SEQ ID NO:2.The inventors have determined that the above polypeptide fragments areantigenic regions of the Human Cortistatin protein. Methods fordetermining other such epitope-bearing portions of the Human Cortistatinprotein are described in detail below.

[0039] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC Deposit No. 97639. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0040] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below.

[0041] Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50-300 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in SEQ ID NO:1. By a portion of a polynucleotide of“at least 20 nt in length,” for example, is intended 20 or morecontiguous nucleotides from the nucleotide sequence of the referencepolynucleotide (e.g., the deposited cDNA or the nucleotide sequence asshown in SEQ ID NO:1). As indicated, such portions are usefuldiagnostically either as a probe according to conventional DNAhybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), the entire disclosureof which is hereby incorporated herein by reference.

[0042] Since a Human Cortistatin cDNA clone has been deposited and itsdetermined nucleotide sequence is provided in SEQ ID NO:1, generatingpolynucleotides which hybridize to a portion of the Human CortistatincDNA molecule would be routine to the skilled artisan. For example,restriction endonuclease cleavage or shearing by sonication of the HumanCortistatin cDNA clone could easily be used to generate DNA portions ofvarious sizes which are polynucleotides that hybridize to a portion ofthe Human Cortistatin cDNA molecule. Alternatively, the hybridizingpolynucleotides of the present invention could be generatedsynthetically according to known techniques. Of course, a polynucleotidewhich hybridizes only to a poly A sequence (such as the 3′ terminalpoly(A) tract of the Human Cortistatin cDNA shown in SEQ ID NO:1), or toa complementary stretch of T (or U) resides, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

[0043] As indicated, nucleic acid molecules of the present inventionwhich encode the Cortistatin protein may include, but are not limited tothose encoding the amino acid sequence of the mature polypeptide, byitself, the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 19 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37:767 (1984).As discussed below, other such fusion proteins include Human Cortistatinfused to Fc at the N- or C-terminus.

[0044] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the Cortistatin protein. Variants may occur naturally,such as a natural allelic variant. By an “allelic variant” is intendedone of several alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley &Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0045] Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the Cortistatin protein or portionsthereof. Also especially preferred in this regard are conservativesubstitutions. Most highly preferred are nucleic acid molecules encodingthe mature protein having the amino acid sequence shown in SEQ ID NO:2or the mature Human Cortistatin amino acid sequence encoded by thedeposited cDNA clone.

[0046] The nucleotide at position 314 in FIG. 1 (SEQ ID NO:1) may beeither an A or a G resulting in the encoded amino acid being either K(lysine) or R (arginine). Similarly, position 342 may be an A or a G(conserved change), and position 348 may be a T or a C (conservedchange).

[0047] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 95% identical, and more preferably at least 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 86 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 58 to about 86 in SEQ ID NO:2; (e) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97639; (f) a nucleotidesequence encoding the mature Human Cortistatin polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97639; or (g) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), or (f).

[0048] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aHuman Cortistatin polypeptide is intended that the nucleotide sequenceof the polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence encoding theHuman Cortistatin polypeptide. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

[0049] As a practical matter, whether any particular nucleic acidmolecule is at least 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in SEQ ID NO:1 or to thenucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0050] The present application is directed to nucleic acid molecules atleast 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in SEQ ID NO:1 or to the nucleic acid sequence of the depositedcDNA, irrespective of whether they encode a polypeptide havingCortistatin activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having Cortistatin activity,one of skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having Cortistatin activityinclude, inter alia, (1) isolating the Cortistatin gene or allelicvariants thereof in a cDNA library; (2) in situ hybridization (e.g.,“FISH”) to metaphase chromosomal spreads to provide precise chromosomallocation of the Cortistatin gene, as described in Verma et al., HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York(1988); and (3) Northern Blot analysis for detecting Cortistatin mRNAexpression in specific tissues (e.g., putamen; cerebral cortex andhippocampus).

[0051] Preferred, however, are nucleic acid molecules having sequencesat least 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in SEQ ID NO:1 or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide havingCortistatin protein activity. By “a polypeptide having Cortistatinactivity” is intended polypeptides exhibiting activity similar, but notnecessarily identical, to an activity of the Cortistatin protein of theinvention (either the full-length protein or, preferably, the matureprotein), as measured in a particular biological assay.

[0052] Human Cortistatin exhibits activity in sleep regulation, forexample, by depressing neuronal electrical activity, inducing lowfrequency cerebral waves in the cerebral cortex, and antagonizing theeffects of acetylcholine in the hippocampus and cortex measures ofexcitability. For example, one can measure Cortistatin activity bymanually depolarizing a CA1 neuron to about −65 mV (resting potential atabout −70 mV) to elicit action potential firing. Superfusion by 1 μMHuman Cortistatin would abolish the action potential discharge. One canmeasure Population Spike (PS) amplitudes in CA1 neurons in vivo, bygenerating stimulus response curves and relating the PS amplitudemonotonically to stimulus intensity tested at three response levels:threshold, half-maximal, and maximal. Microiontophoretic applications of1 mg ml⁻¹ of Cortistatin or a polypeptide having Cortistatin activitywould significantly decrease PS amplitudes in CA1. Another assay forpolypeptides having Cortistatin activity is the effect ofintracerebroventricular administration to male Sprague—Dawley rats,anesthetized with halothane, of the polypeptide in question on thesleep—wake cycle of the rat. An active polypeptide would increase theperiod of slow wave sleep and decrease REM sleep. Yet another assay isthe inhibition effect by the Cortistatin activity-exhibiting polypeptideon local Electroencephalographic Activity (EEG) in the visual cortexwhich would otherwise be seen by administration of acetyl choline (ACh).Microiontophoretic application of Cortistatin and ACh, or of Cortistatinalone will not increase the averaged EEG power spectra in the 8-16 Hzfrequency range, whereas administration of ACh alone will markedlyincrease such power spectra.

[0053] Thus, “a polypeptide having Cortistatin protein activity”includes polypeptides that also exhibit any of the same neuropeptidemodulating activities in the above-described assays in a dose-dependentmanner. Although the degree of dose-dependent activity need not beidentical to that of the Human Cortistatin protein, preferably, “apolypeptide having Cortistatin protein activity” will exhibitsubstantially similar dose-dependence in a given activity as compared tothe Cortistatin protein (i.e., the candidate polypeptide will exhibitgreater activity or not more than about tenfold less and, preferably,not more than about twofold less activity relative to the referenceHuman Cortistatin protein). One polypeptide believed to have Cortistatinprotein activity is the 29 amino acid sequence shown in FIG. 4 (SEQ IDNO:4) (amino acids from about 58 to about 86 in SEQ ID NO:2).

[0054] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 95%, 97%, 98%,or 99% identical to the nucleic acid sequence of the deposited cDNA orthe nucleic acid sequence shown in SEQ ID NO:1 will encode “apolypeptide having Cortistatin protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving Cortistatin protein activity. This is because the skilled artisanis fully aware of amino acid substitutions that are either less likelyor not likely to significantly effect protein function (e.g., replacingone aliphatic amino acid with a second aliphatic amino acid).

[0055] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U., et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main approaches for studying the toleranceof an amino acid sequence to change. The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U., et al., supra, and thereferences cited therein.

[0056] Vectors and Host Cells

[0057] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof Cortistatin polypeptides or fragments thereof by recombinanttechniques.

[0058] Recombinant constructs may be introduced into host cells usingwell known techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retrovirat vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0059] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0060] Preferred are vectors comprising cis-acting control regions tothe polynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

[0061] In certain preferred embodiments in this regard, the vectorsprovide for specific expression, which may be inducible and/or celltype-specific. Particularly preferred among such vectors are thoseinducible by environmental factors that are easy to manipulate, such astemperature and nutrient additives.

[0062] Expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, bacteriophage, yeast episomes, yeastchromosomal elements, viruses such as baculoviruses, papova viruses,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

[0063] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

[0064] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

[0065] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0066] Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

[0067] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0068] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0069] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0070] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16:9459-9471 (1995).

[0071] The Cortistatin protein can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0072] Cortistatin Polypeptides and Fragments

[0073] The invention further provides an isolated Cortistatinpolypeptide having the amino acid sequence encoded by the depositedcDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide orpolypeptide comprising a portion of the above polypeptides. The terms“peptide” and “oligopeptide” are considered synonymous (as is commonlyrecognized) and each term can be used interchangeably as the contextrequires to indicate a chain of at least to amino acids coupled bypeptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than ten amino acid residues. All oligopeptide andpolypeptide formulas or sequences herein are written from left to rightand in the direction from amino terminus to carboxy terminus.

[0074] It will be recognized in the art that some amino acid sequencesof the Cortistatin polypeptide can be varied without significant effectof the structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity. In general, itis possible to replace residues which form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

[0075] Thus, the invention further includes variations of theCortistatin polypeptide which show substantial Cortistatin polypeptideactivity or which include regions of Cortistatin protein such as theprotein portions discussed below. Such mutants include deletions,insertions, inversions, repeats, and type substitutions (for example,substituting one hydrophilic residue for another, but not stronglyhydrophilic for strongly hydrophobic as a rule). Small changes or such“neutral” amino acid substitutions will generally have little effect onactivity.

[0076] Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu, substitution between the amideresidues Asn and Gln, exchange of the basic residues Lys and Arg andreplacements among the aromatic residues Phe, Tyr.

[0077] As indicated in detail above, further guidance concerning whichamino acid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

[0078] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2, or that encoded by the deposited cDNA, may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

[0079] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the Cortistatin protein. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 10:307-377 (1993)).

[0080] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0081] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove and below.

[0082] Generally speaking, the number of substitutions for any givenCortistatin polypeptide, or mutant thereof, will not be more than 50,40, 30, 20, 10, 5, or 3, depending on the objective.

[0083] Amino acids in the Cortistatin protein of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity using the assays described supra. Sites that arecritical for ligand-receptor binding can also be determined bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904(1992) and de Vos et al. Science 255:306-312 (1992)).

[0084] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of the Human Cortistatin polypeptide canbe substantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0085] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of the Cortistatin polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0086] The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader, the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein), a polypeptide comprising amino acids about −19 toabout 86 in SEQ ID NO:2; a polypeptide comprising amino acids about −18to about 86 in SEQ ID NO:2; a polypeptide comprising amino acids about 1to about 86 in SEQ ID NO:2; a polypeptide comprising amino acids about58 to about 86 in SEQ ID NO:2; as well as polypeptides which are atleast 95% identical, more preferably at least 96% or 97% identical,still more preferably at least 98% or 99% identical to those describedabove and also include portions of such polypeptides with at least 30amino acids and more preferably at least 50 amino acids.

[0087] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a HumanCortistatin polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the HumanCortistatin polypeptide. In other words, to obtain a polypeptide havingan amino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0088] As a practical matter, whether any particular polypeptide is atleast 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby the deposited cDNA clone can be determined conventionally using knowncomputer programs such the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

[0089] The polypeptide of the present invention could be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0090] As described in detail below, the polypeptides of the presentinvention can also be used to raise polyclonal and monoclonalantibodies, which are useful in assays for detecting Cortistatin proteinexpression as described below or as agonists and antagonists capable ofenhancing or inhibiting Cortistatin protein function. Further, suchpolypeptides can be used in the yeast two-hybrid system to “capture”Cortistatin protein binding proteins which are also candidate agonistand antagonist according to the present invention. The yeast two hybridsystem is described in Fields and Song, Nature 340:245-246 (1989).

[0091] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide of the invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. These immunogenicepitopes are believed to be confined to a few loci on the molecule. Onthe other hand, a region of a protein molecule to which an antibody canbind is defined as an “antigenic epitope.” The number of immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983).

[0092] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies thatreact with predetermined sites on proteins. Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Peptides that are extremelyhydrophobic and those of six or fewer residues generally are ineffectiveat inducing antibodies that bind to the mimicked protein; longer,peptides, especially those containing proline residues, usually areeffective. Sutcliffe et al., supra, at 661. For instance, 18 of 20peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

[0093] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.Thus, a high proportion of hybridomas obtained by fusion of spleen cellsfrom donors immunized with an antigen epitope-bearing peptide generallysecrete antibody reactive with the native protein. Sutcliffe et al,supra, at 663. The antibodies raised by antigenic epitope-bearingpeptides or polypeptides are useful to detect the mimicked protein, andantibodies to different peptides may be used for tracking the fate ofvarious regions of a protein precursor which undergoespost-translational processing. The peptides and anti-peptide antibodiesmay be used in a variety of qualitative or quantitative assays for themimicked protein, for instance in competition assays since it has beenshown that even short peptides (e.g., about 9 amino acids) can bind anddisplace the larger peptides in immunoprecipitation assays. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

[0094] Antigenic epitope-bearing peptides and polypeptides of theinvention designed according to the above guidelines preferably containa sequence of at least seven, more preferably at least nine and mostpreferably between about 15 to about 30 amino acids contained within theamino acid sequence of a polypeptide of the invention. However, peptidesor polypeptides comprising a larger portion of an amino acid sequence ofa polypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

[0095] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate Cortistatin-specific antibodies include: apolypeptide comprising amino acid residues from about 4 to about 19 inSEQ ID NO:2; a polypeptide comprising amino acid residues from about 21to about 28 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 39 to about 46 in SEQ ID NO:2; a polypeptide comprising aminoacid residues from about 47 to about 79 in SEQ ID NO:2; and apolypeptide comprising amino acid residues from about 81 to about 86 inSEQ ID NO:2. As indicated above, the inventors have determined that theabove polypeptide fragments are antigenic regions of the Cortistatinprotein.

[0096] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means for making peptides orpolypeptides including recombinant means using nucleic acid molecules ofthe invention. For instance, a short epitope-bearing amino acid sequencemay be fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500-1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

[0097] Epitope-bearing peptides and polypeptides of the invention areused to induce antibodies according to methods well known in the art.See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow,M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. etal., J. Gen. Virol. 66:2347-2354 (1985). Generally, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling of the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine may be coupled to carrier using a linkersuch as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

[0098] Immunogenic epitope-bearing peptides of the invention, i.e.,those parts of a protein that elicit an antibody response when the wholeprotein is the immunogen, are identified according to methods known inthe art. For instance, Geysen et al., supra, discloses a procedure forrapid concurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al supra with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

[0099] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

[0100] The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

[0101] As one of skill in the art will appreciate, Cortistatinpolypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric Cortistatin protein orprotein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)).

[0102] Neurological Disorder Diagnosis

[0103] Cortistatin is expressed primarily in the putamen. For a numberof neurological, sleep-related disorders, substantially altered(increased or decreased) levels of Cortistatin gene expression can bedetected in brain tissue or other cells or bodily fluids (e.g., sera,plasma, urine, synovial fluid or spinal fluid) taken from an individualhaving such a disorder, relative to a “standard” Cortistatin geneexpression level, that is, the Cortistatin expression level in tissue orbodily fluids from an individual not having the disorder. Thus, theinvention provides a diagnostic method useful during diagnosis of asleep disorder, which involves measuring the expression level of thegene encoding the Cortistatin protein in brain tissue or other cells orbody fluid from an individual and comparing the measured gene expressionlevel with a standard Cortistatin gene expression level, whereby anincrease or decrease in the gene expression level compared to thestandard is indicative of such a disorder.

[0104] By individual is intended mammalian individuals, preferablyhumans. By “measuring the expression level of the gene encoding theCortistatin protein” is intended qualitatively or quantitativelymeasuring or estimating the level of the Cortistatin protein or thelevel of the mRNA encoding the Cortistatin protein in a first biologicalsample either directly (e.g., by determining or estimating absoluteprotein level or mRNA level) or relatively (e.g., by comparing to theCortistatin protein level or mRNA level in a second biological sample).Preferably, the Cortistatin protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardCortistatin protein level or mRNA level, the standard being taken from asecond biological sample obtained from an individual not having thedisorder or being determined by averaging levels from a population ofindividuals not having a neurological sleep disorder. As will beappreciated in the art, once a standard Cortistatin protein level ormRNA level is known, it can be used repeatedly as a standard forcomparison.

[0105] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains Cortistatin protein or mRNA. As indicated,biological samples include body fluids (such as sera, plasma, urine,synovial fluid and spinal fluid) which contain secreted matureCortistatin protein, brain tissue, and other tissue sources found toexpress Cortistatin or a Cortistatin receptor. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

[0106] The present invention is useful for diagnosis or treatment ofvarious neurological-related disorders in mammals, preferably humans.Such disorders include the following tumors and cancers, hypoactivity,hyperactivity, atrophy, enlargement of the putamen, and the like.

[0107] Total cellular RNA can be isolated from a biological sample usingany suitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the Cortistatin protein are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

[0108] Northern blot analysis can be performed as described in Harada etal., Cell 63:303-312 (1990). Briefly, total RNA is prepared from abiological sample as described above. For the Northern blot, the RNA isdenatured in an appropriate buffer (such as glyoxal/dimethylsulfoxide/sodium phosphate buffer), subjected to agarose gelelectrophoresis, and transferred onto a nitrocellulose filter. After theRNAs have been linked to the filter by a UV linker, the filter isprehybridized in a solution containing formamide, SSC, Denhardt'ssolution, denatured salmon sperm, SDS, and sodium phosphate buffer.Cortistatin protein cDNA labeled according to any appropriate method(such as the ³²P-multiprimed DNA labeling system (Amersham)) is used asprobe. After hybridization overnight, the filter is washed and exposedto x-ray film. cDNA for use as probe according to the present inventionis described in the sections above and will preferably at least 15 bp inlength.

[0109] S1 mapping can be performed as described in Fujita et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe Cortistatin protein). Northern blot analysis can be performed asdescribed above.

[0110] Preferably, levels of mRNA encoding the Cortistatin protein areassayed using the RT-PCR method described in Makino et al., Technique2.295-301 (1990). By this method, the radioactivities of the “amplicons”in the polyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the HumanCortistatin protein) is quantified using an imaging analyzer. RT and PCRreaction ingredients and conditions, reagent and gel concentrations, andlabeling methods are well known in the art. Variations on the RT-PCRmethod will be apparent to the skilled artisan.

[0111] Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

[0112] Assaying Cortistatin protein levels in a biological sample canoccur using any art-known method. Preferred for assaying Cortistatinprotein levels in a biological sample are antibody-based techniques. Forexample, Cortistatin protein expression in tissues can be studied withclassical immunohistological methods. In these, the specific recognitionis provided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of Cortistatin protein for Western-blot or dot/slotassay (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985));Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). In thistechnique, which is based on the use of cationic solid phases,quantitation of Cortistatin protein can be accomplished using isolatedCortistatin protein as a standard. This technique can also be applied tobody fluids. With these samples, a molar concentration of Cortistatinprotein will aid to set standard values of Cortistatin protein contentfor different body fluids, like serum, plasma, urine, synovial fluid,spinal fluid, etc. The normal appearance of Cortistatin protein amountscan then be set using values from healthy individuals, which can becompared to those obtained from a test subject.

[0113] Other antibody-based methods useful for detecting Cortistatinprotein levels include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example,Human Cortistatin protein-specific monoclonal antibodies can be usedboth as an immunoadsorbent and as an enzyme-labeled probe to detect andquantify, the Cortistatin protein. The amount of Cortistatin proteinpresent in the sample can be calculated by reference to the amountpresent in a standard preparation using a linear regression computeralgorithm. Such an ELISA for detecting a tumor antigen is described inIacobelli et al., Breast Cancer Research and Treatment 11:19-30 (1988).In another ELISA assay, two distinct specific monoclonal antibodies canbe used to detect Cortistatin protein in a body fluid. In this assay,one of the antibodies is used as the immunoadsorbent and the other asthe enzyme-labeled probe.

[0114] The above techniques may be conducted essentially as a “one-step”or “two-step” assay. The “one-step” assay involves contactingCortistatin protein with immobilized antibody and, without washing,contacting the mixture with the labeled antibody. The “two-step” assayinvolves washing before contacting the mixture with the labeledantibody. Other conventional methods may also be employed as suitable.It is usually desirable to immobilize one component of the assay systemon a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed from thesample.

[0115] Suitable enzyme labels include, for example, those from theoxidase group, which catalyze the production of hydrogen peroxide byreacting with substrate. Glucose oxidase is particularly preferred as ithas good stability and its substrate (glucose) is readily available.Activity of an oxidase label may be assayed by measuring theconcentration of hydrogen peroxide formed by the enzyme-labeledantibody/substrate reaction. Besides enzymes, other suitable labelsinclude radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0116] In addition to assaying Cortistatin protein levels in abiological sample obtained from an individual, Cortistatin protein canalso be detected in vivo by imaging. Antibody labels or markers for invivo imaging of Cortistatin protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0117] A Cortistatin protein-specific antibody or antibody portion whichhas been labeled with an appropriate detectable imaging moiety, such asa radioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a disorderinvolving the hippocampus and/or the cerebral cortex regions of thebrain. It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietiesneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of ^(99m)Tc. The labeledantibody or antibody portion will then preferentially accumulate at thelocation of cells which contain Cortistatin protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Portions” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, eds., S. W. Burchiel andB. A. Rhodes, Masson Publishing Inc. (1982)).

[0118] Cortistatin-protein specific antibodies for use in the presentinvention can be raised against the intact Cortistatin protein or anantigenic polypeptide portion thereof, which may presented together witha carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier.

[0119] As used herein, the term “antibody” (Ab) or “monoclonal antibody”(Mab) is meant to include intact molecules as well as antibody portions(such as, for example, Fab and F(ab′)₂ portions) which are capable ofspecifically binding to Human Cortistatin protein. Fab and F(ab′)₂portions lack the Fc portion of intact antibody, clear more rapidly fromthe circulation, and may have less non-specific tissue binding of anintact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus,these portions are preferred.

[0120] The antibodies of the present invention may be prepared by any ofa variety of methods. For example, cells expressing the Cortistatinprotein or an antigenic portion thereof can be administered to an animalin order to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of Cortistatin proteinis prepared and purified as described above to render it substantiallyfree of natural contaminants. Such a preparation is then introduced intoan animal in order to produce polyclonal antisera of greater specificactivity.

[0121] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or Cortistatin protein bindingportions thereof). Such monoclonal antibodies can be prepared usinghybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler etal., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J Immunol. 6:292(1976); Hammerling et al., In: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, suchprocedures involve immunizing an animal (preferably a mouse) with aCortistatin protein antigen or, more preferably with a Cortistatinprotein-expressing cell. Suitable cells can be recognized by theircapacity to bind anti-Cortistatin protein antibody. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 μg/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP₂O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232(1981)). The hybridoma cells obtained through such a selectionare then assayed to identify clones which secrete antibodies capable ofbinding the Cortistatin antigen.

[0122] Alternatively, additional antibodies capable of binding to theCortistatin protein antigen may be produced in a two-step procedurethrough the use of anti-idiotypic antibodies. Such a method inakes useof the fact that antibodies are themselves antigens, and therefore it ispossible to obtain an antibody which binds to a second antibody. Inaccordance with this method, Cortistatin protein specific antibodies areused to immunize an animal, preferably a mouse. The splenocytes of suchan animal are then used to produce hybridoma cells, and the hybridomacells are screened to identify clones which produce an antibody whoseability to bind to the Cortistatin protein-specific antibody can beblocked by the Cortistatin protein antigen. Such antibodies compriseanti-idiotypic antibodies to the Cortistatin protein-specific antibodyand can be used to immunize an animal to induce formation of furtherCortistatin protein-specific antibodies.

[0123] It will be appreciated that Fab and F(ab′)₂ and other portions ofthe antibodies of the present invention may be used according to themethods disclosed herein. Such portions are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabportions) or pepsin (to produce F(ab′)₂ portions). Alternatively,Cortistatin protein-binding portions can be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

[0124] Where in vivo imaging is used to detect enhanced levels ofCortistatin protein for diagnosis in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229.1202(1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314.268 (1985).

[0125] Additionally, it is also possible to use so called Single ChainAntibodies, which contain the variable region of a light chain, thevariable region of a heavy chain, both of these variable regions fusedvia a peptide linker. Such antibodies are described, for example inLadner et al. U.S. Pat. No. 4,946,778.

[0126] Any of the antibodies of the invention can be either mono orbispecific.

[0127] Further suitable labels for the Cortistatin protein-specificantibodies of the present invention are provided below. Examples ofsuitable enzyme labels include malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

[0128] Examples of suitable radioisotopic labels include ³H, ¹¹¹In,¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y,⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotopewhere in vivo imaging is used since its avoids the problem ofdehalogenation of the ¹²⁵I or ¹³¹I-labeled monoclonal antibody by theliver. In addition, this radionucleotide has a more favorable gammaemission energy for imaging (Perkins et al., Eur. J. Nucl. Med.10:296-301 (1985); Carasquillo et al., J. Nucl. Med. 28:281-287(1987)).For example, ¹¹¹In coupled to monoclonal antibodies with1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumoroustissues, particularly the liver, and therefore enhances specificity oftumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)).

[0129] Examples of suitable non-radioactive isotopic labels include¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

[0130] Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

[0131] Examples of suitable toxin labels include diphtheria toxin,ricin, and cholera toxin.

[0132] Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

[0133] Examples of nuclear magnetic resonance contrasting agents includeheavy metal nuclei such as Gd, Mn, and Fe.

[0134] Typical techniques for binding the above-described labels toantibodies are provided by Kennedy et al (Clin. Chim. Acta 70:1-31(1976)), and Schurs et al. (Clin. Chim. Acta 81:1-40 (1977)). Couplingtechniques mentioned in the latter are the glutaraldehyde method, theperiodate method, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

[0135] Chromosome Assays

[0136] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

[0137] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a Cortistatin protein gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose. Typically, in accordance with routine procedures forchromosome mapping, some trial and error may be necessary to identify agenomic probe that gives a good in situ hybridization signal.

[0138] In some cases, in addition, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedportion.

[0139] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of portions from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0140] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES,Pergamon Press, New York (1988).

[0141] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, MENDELIAN INHERITANCE IN MAN, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0142] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0143] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0144] Treatment of Sleep-Related and Other Neurological Disorders

[0145] It will be appreciated by one of ordinary skill that, since theCortistatin protein of the invention is translated with a leader peptidesuitable for secretion of the mature protein from the cells whichexpress Cortistatin, when Cortistatin protein (particularly the matureform) is added from an exogenous source to cells, tissues or the body ofan individual, the protein will exert its modulating activities on anyof its target cells of that individual. Therefore, it will beappreciated that conditions caused by a decrease in the standard ornormal level of Cortistatin activity in an individual, particularlysleep disorders, can be treated be administration of Cortistatinprotein. Thus, the invention also provides a method of treatment of anindividual in need of an increased level of Cortistatin activitycomprising administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated Cortistatin polypeptideof the invention, particularly a mature form of the Cortistatin proteinof the invention, effective to increase the Cortistatin activity levelin such an individual.

[0146] Such conditions are generally those related to regulatingneuronal activity in an individual, especially sleep. The Cortistatin ofthe invention antagonizes the effects of ACh in both the hippocampus andthe cortex, and is to administered when such effects are desired.

[0147] One of ordinary skill will appreciate that effective amounts ofthe Cortistatin polypeptides for treating an individual in need of anincreased level of Cortistatin activity (including amounts ofCortistatin polypeptides effective for sleep regulation) can bedetermined empirically for each condition where administration ofCortistatin is indicated. The polypeptide having Cortistatin activity mybe administered in pharmaceutical compositions in combination with oneor more pharmaceutically acceptable excipients. It will be understoodthat, when administered to a human patient, the total daily usage of thepharmaceutical compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the type and degree ofthe response to be achieved; the specific composition an other agent, ifany, employed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the composition; the duration of the treatment; drugs(such as a chemotherapeutic agent) used in combination or coincidentalwith the specific composition; and like factors well known in themedical arts.

[0148] The Cortistatin composition to be used in the therapy will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with Cortistatin alone), thesite of delivery of the Cortistatin composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of Cortistatin forpurposes herein (including a Cortistatin effective amount) is thusdetermined by such considerations.

[0149] As a general proposition, the total pharmaceutically effectiveamount of the Cortistatin administered parenterally per dose will be inthe range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Cortistatin is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The key factor in selecting anappropriate dose is the result obtained, as measured by decreases inneuronal activity, sleep alterations, etc.

[0150] A course of Cortistatin treatment to affect the neurologicalsystem appears to be optimal if continued longer than a certain minimumnumber of days. The length of treatment needed to observe changes andthe interval following treatment for responses to occur appears to varydepending on the desired effect.

[0151] The Cortistatin is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman etal., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release Cortistatin compositions also include liposomallyentrapped Cortistatin. Liposomes containing Cortistatin are prepared bymethods known per se: DE 3,218,121; Epstein, et al., Proc. Natl. Acad.Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal Cortistatin therapy.

[0152] For parenteral administration, in one embodiment, the Cortistatinis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

[0153] Generally, the formulations are prepared by contacting theCortistatin uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0154] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0155] The Cortistatin is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of Cortistatin salts.

[0156] Cortistatin to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). TherapeuticCortistatin compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

[0157] Cortistatin ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampules or vials, as an aqueous solutionor as a lyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Cortistatin solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Cortistatin using bacteriostaticWater-for-Injection.

[0158] The active polypeptide can also be administered in the form ofliposomes. As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to theagent or inhibitor, stabilizers, preservatives, excipients, and thelike. The preferred lipids are the phospholipids and the phosphatidylcholates (lecithins), both natural and synthetic. Methods to formliposomes are known in the art. See, for example, Prescott, Ed., Methodsin Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p.33 et seq.

[0159] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Cloning and Expression of Human Cortistatin in aBaculovirus Expression System

[0160] In this illustrative example, the plasmid shuttle vector pA2 isused to insert the cloned DNA encoding the complete protein, includingits naturally associated secretary signal (leader) sequence, into abaculovirus to express the mature Human Cortistatin protein, usingstandard methods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

[0161] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

[0162] The cDNA sequence encoding the full length Human Cortistatinprotein in the deposited clone, including the AUG initiation codon andthe naturally associated leader sequence shown in FIG. 1 (SEQ ID NO:2),is amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene. The 5′ primer has the sequence: 5′GACTGGATCCGCCATCATGCCATTGTCCCCCGGCC 3′ (SEQ ID NO:5) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 19 bases of thesequence of the complete Human Cortistatin protein shown in FIG. 1,beginning with the AUG initiation codon. The 3′ primer has the sequence:5′GACTGGTACCGGTCTGTCATTACACTTGC 3′(SEQ ID NO:6) containing theunderlined, Asp718 restriction site followed by 17 nucleotidescomplementary to the 3′ noncoding sequence in FIG. 1.

[0163] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein“F1”.

[0164] The plasmid is digested with the restriction enzymes BamHI andAsp718 and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1 ”.

[0165] Fragment F1 and the dephosphorylated plasmid VI are ligatedtogether with T4 DNA ligase. E. Coli HB 101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with the HumanCortistatin gene using the PCR method, in which one of the primers thatis used to amplify the gene and the second primer is from well withinthe vector so that only those bacterial colonies containing the HumanCortistatin gene fragment will show amplification of the DNA. Thesequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pBacCortistatin.

[0166] Five μg of the plasmid pBacCortistatin is co-transfected with 1.0μg of a commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacCortistatin are mixed in a sterile well of a microtiterplatecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

[0167] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-Cortistatin.

[0168] To verify the expression of the Human Cortistatin gene, Sf9 cellsare grown in Grace's medium supplemented with 10% heat inactivated FBS.The cells are infected with the recombinant baculovirus V-Cortistatin ata multiplicity of infection (“MOI”) of about 2. Six hours later themedium is removed and is replaced with SF900 II medium minus methionineand cysteine (available from Life Technologies Inc., Rockville, Md.). Ifradiolabeled proteins are desired, 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled). Microsequencing of the amino acidsequence of the amino terminus of purified protein may be used todetermine the amino terminal sequence of the mature protein and thus thecleavage point and length of the secretory signal peptide.

Example 2 Expression and Purification of Human Cortistatin in E. coli

[0169] The bacterial expression vector pQE60 is used for bacterialexpression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE60 encodes ampicillin antibiotic resistance (“Ampr”)and contains a bacterial origin of replication (“ori”), an IPTGinducible promoter, a ribosome binding site (“RBS”), six codons encodinghistidine residues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6×His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6×His tag.

[0170] The DNA sequence encoding the desired portion of the HumanCortistatin protein lacking the hydrophobic leader sequence is amplifiedfrom the deposited cDNA clone using PCR oligonucleotide primers whichanneal to the amino terminal sequences of the desired portion of theHuman Cortistatin protein and to sequences in the deposited construct 3′to the cDNA coding sequence. Additional nucleotides containingrestriction sites to facilitate cloning in the pQE60 vector are added tothe 5′ and 3′ sequences, respectively.

[0171] For cloning the mature protein, the 5′ primer has the sequence:5′ GACTCCATGGCCCTGCCCCTGGAGG 3′ (SEQ ID NO:7) containing the underlinedNcoI restriction site followed by 15 nucleotides complementary to theamino terminal coding sequence of the mature Human Cortistatin sequencein FIG. 1. One of ordinary skill in the art would appreciate, of course,that the point in the protein coding sequence where the 5′ primer beginsmay be varied to amplify a desired portion of the complete proteinshorter or longer than the mature form. The 3′ primer has the sequence:5′ AGATCTFTGCAGGAGGAGAAGG 3′ (SEQ ID NO:8) containing the underlinedBglII restriction site followed by 17 nucleotides complementary to the3′ end of the protein coding sequence in the Human Cortistatin DNAsequence in FIG. 1.

[0172] The amplified Human Cortistatin DNA fragments and the vectorpQE60 are digested with NcoI and BglII and the digested DNAs are thenligated together. Insertion of the Human Cortistatin DNA into therestricted pQE60 vector places the Human Cortistatin protein codingregion including its associated stop codon downstream from theIPTG-inducible promoter and in-frame with an initiating AUG. Theassociated stop codon prevents translation of the six histidine codonsdownstream of the insertion point.

[0173] The ligation mixture is transformed into competent E. coli cellsusing standard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing Human Cortistatin protein, is available commercially fromQIAGEN, Inc., supra. Transformants are identified by their ability togrow on LB plates in the presence of ampicillin and kanamycin. PlasmidDNA is isolated from resistant colonies and the identity of the clonedDNA confirmed by restriction analysis, PCR and DNA sequencing.

[0174] Clones containing the desired constructs are grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used toinoculate a large culture, at a dilution of approximately 1:25 to 1:250.The cells are grown to an optical density at 600 nm (“OD600”) of between0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then addedto a final concentration of 1 mM to induce transcription from the lacrepressor sensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

[0175] The cells are then stirred for 3-4 hours at 4° C. in 6Mguanidine-HCl, pH 8. The cell debris is removed by centrifugation, andthe supernatant containing the Human Cortistatin is dialyzed against 50mM Na-acetate buffer pH 6, supplemented with 200 mM NaCl. Alternatively,the protein can be successfully refolded by dialyzing it against 500 mMNaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4, containing proteaseinhibitors. After renaturation the protein can be purified by ionexchange, hydrophobic interaction and size exclusion chromatography.Alternatively, an affinity chromatography step such as an antibodycolumn can be used to obtain pure Human Cortistatin protein. Thepurified protein is stored at 4° C. or frozen at −80° C.

Example 3 Cloning and Expression of Human Cortistatin in Mammalian Cells

[0176] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human HeLa 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0177] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, or hygromycinallows the identification and isolation of the transfected cells.

[0178] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of proteins.

[0179] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

[0180] The expression plasmid, pCoitistatin HA, is made by cloning acDNA encoding Human Cortistatin into the expression vector pcDNAI/Amp orpcDNAIII (which can be obtained from Invitrogen, Inc.).

[0181] The expression vector pcDNAI/amp contains: (I) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0182] A DNA fragment encoding the Human Cortistatin protein is clonedinto the polylinker region of the vector so that recombinant proteinexpression is directed by the CMV promoter. The plasmid constructionstrategy is as follows. The Human Cortistatin cDNA of the depositedclone is amplified using primers that contain convenient restrictionsites, much as described above for construction of vectors forexpression of Human Cortistatin in E. coli. Suitable primers include thefollowing, which are used in this example. The 5′ primer, containing theunderlined BamHI site, a Kozak sequence, an AUG start codon and 19nucleotides of the 5′ coding region of the complete Human Cortistatincoding sequence has the following sequence: 5′GACTGGATCCGCCATCATGCCATTGTCCCCCGGCC 3′ (SEQ ID NO:5).

[0183] The 3′ primer, containing the underlined Asp718 site, a stopcodon, and 17 bp of 3′ non-coding sequence has the following sequence:5′ GACTGGTACCGGTCTGTCATTACACTTGC 3′ (SEQ ID NO:6).

[0184] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with BamHI and Asp718 and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), andthe—transformed culture is plated on ampicillin media plates which thenare incubated to allow growth of ampicillin resistant colonies. PlasmidDNA is isolated from resistant colonies and examined by restrictionanalysis or other means for the presence of the HumanCortistatin-encoding fragment.

[0185] For expression of recombinant Human Cortistatin, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989). Cells are incubated under conditions for expressionof Human Cortistatin by the vector.

[0186] Expression of the Human Cortistatin-HA fusion protein is detectedby radiolabeling and immunoprecipitation, using methods described in,for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). Tothis end, two days after transfection, the cells are labeled byincubation in media containing ³⁵S-cysteine for 8 hours. The cells andthe media are collected, and the cells are washed and lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5%DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.Proteins are precipitated from the cell lysate and from the culturemedia using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

[0187] The vector pC4 is used for the expression of Human Cortistatinprotein. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCCAccession No. 37146). The plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary- or othercells lacking dihydrofolate activity that are transfected with theseplasmids can be selected by growing the cells in a selective medium(alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,1978, J Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990,Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A.1991, Biotechnology 9:64-68). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

[0188] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-447) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI, andAsp718 restriction enzyme cleavage sites that allow integration of thegenes. Behind these cloning sites the plasmid contains the 3′ intron andpolyadenylation site of the rat preproinsulin gene. Other highefficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the Human Cortistatin in a regulated wayin mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89: 5547-5551). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0189] The plasmid pC4 is digested with the restriction enzymes BamHIand Asp718 and then dephosphorylated using calf intestinal phosphataseby procedures known in the art. The vector is then isolated from a 1%agarose gel.

[0190] The DNA sequence encoding the complete Human Cortistatin proteinincluding its leader sequence is amplified using PCR oligonucleotideprimers corresponding to the 5′ and 3′ sequences of the gene. The 5′primer has the sequence: 5′ GACTGGATCCGCCATCATGCCATTGTCCCCCGGCC 3′ (SEQID NO:5) containing the underlined BamHI restriction enzyme sitefollowed by an efficient signal for initiation of translation ineukaryotes, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987),and 19 bases of the coding sequence of Human Cortistatin shown in FIG. 1(SEQ ID NO:1). The 3′ primer has the sequence: 5′GACTGGTACCGGTCTGTCATTACACTTGC 3′ (SEQ ID NO:6) containing the underlinedAsp718 restriction site followed by 17 nucleotides complementary to thenon-translated region of the Human Cortistatin gene shown in FIG. 1 (SEQID NO:1).

[0191] The amplified fragment is digested with the endonucleases BamHIand Asp718 and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

[0192] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al.,supra). The plasmid pSV2neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4 Tissue Distribution of Human Cortistatin mRNA Expression

[0193] Northern blot analysis is carried out to examine HumanCortistatin gene expression in human tissues, using methods describedby, among others, Sambrook et al., cited above. A cDNA probe containingthe entire nucleotide sequence of the Human Cortistatin protein (SEQ IDNO:1) is labeled with ³²P using the rediprime™ DNA labeling system(Amersham Life Science), according to manufacturer's instructions. Afterlabeling, the probe is purified using a CHROMA SPIN-100™ column(Clontech Laboratories, Inc.), according to manufacturer's protocolnumber PT 1200-1. The purified labeled probe is then used to examinevarious human tissues for Human Cortistatin mRNA.

[0194] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) are obtained fromClontech and are examined with the labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures.

[0195] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0196] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0197] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 14 1 426 DNA Human Cortistatin CDS (46)..(360) sig_peptide (46)..(102)mat_peptide (103)..(360) 1 aagagcagca gcagggtggg agagaagctc cagtcagcccacaag atg cca ttg tcc 57 Met Pro Leu Ser ccc ggc ctc ctg ctg ctg ctg ctctcc ggg gcc acg gcc acc gct gcc 105 Pro Gly Leu Leu Leu Leu Leu Leu SerGly Ala Thr Ala Thr Ala Ala -15 -10 -5 -1 1 ctg ccc ctg gag ggt ggc cccacc ggc cga gac agc gag cat atg cag 153 Leu Pro Leu Glu Gly Gly Pro ThrGly Arg Asp Ser Glu His Met Gln 5 10 15 gaa gcg gca gga ata agg aaa agcagc ctc ctg act ttc ctc gct tgg 201 Glu Ala Ala Gly Ile Arg Lys Ser SerLeu Leu Thr Phe Leu Ala Trp 20 25 30 tgg ttt gag tgg acc tcc cag gcc agtgcc ggg ccc ctc ata gga gag 249 Trp Phe Glu Trp Thr Ser Gln Ala Ser AlaGly Pro Leu Ile Gly Glu 35 40 45 gaa gcc cgg gag gtg gcc agg cgg cag gaaggc gca ccc ccc cag caa 297 Glu Ala Arg Glu Val Ala Arg Arg Gln Glu GlyAla Pro Pro Gln Gln 50 55 60 65 tct gcg cgc cgg gac aga atg ccc tgc aggaac ttc ttc tgg aaa acc 345 Ser Ala Arg Arg Asp Arg Met Pro Cys Arg AsnPhe Phe Trp Lys Thr 70 75 80 ttc tcc tcc tgc aaa taaaacctca cccatgaatgctcacgcaag tgtaatgaca 400 Phe Ser Ser Cys Lys 85 gacctgaata aaatgtattaagcagc 426 2 105 PRT Human Cortistatin 2 Met Pro Leu Ser Pro Gly Leu LeuLeu Leu Leu Leu Ser Gly Ala Thr -15 -10 -5 Ala Thr Ala Ala Leu Pro LeuGlu Gly Gly Pro Thr Gly Arg Asp Ser -1 1 5 10 Glu His Met Gln Glu AlaAla Gly Ile Arg Lys Ser Ser Leu Leu Thr 15 20 25 Phe Leu Ala Trp Trp PheGlu Trp Thr Ser Gln Ala Ser Ala Gly Pro 30 35 40 45 Leu Ile Gly Glu GluAla Arg Glu Val Ala Arg Arg Gln Glu Gly Ala 50 55 60 Pro Pro Gln Gln SerAla Arg Arg Asp Arg Met Pro Cys Arg Asn Phe 65 70 75 Phe Trp Lys Thr PheSer Ser Cys Lys 80 85 3 112 PRT Rat Cortistatin 3 Met Gly Gly Cys SerThr Arg Gly Lys Arg Pro Ser Ala Leu Ser Leu 1 5 10 15 Leu Leu Leu LeuLeu Leu Ser Gly Ile Ala Ala Ser Ala Leu Pro Leu 20 25 30 Glu Ser Gly ProThr Gly Gln Asp Ser Val Gln Asp Ala Thr Gly Gly 35 40 45 Arg Arg Thr GlyLeu Leu Thr Phe Leu Ala Trp Trp His Glu Trp Ala 50 55 60 Ser Gln Asp SerSer Ser Thr Ala Phe Glu Gly Gly Thr Pro Glu Leu 65 70 75 80 Ser Lys ArgGln Glu Arg Pro Pro Leu Gln Gln Pro Pro His Arg Asp 85 90 95 Lys Lys ProCys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 100 105 110 4 29 PRTSynthetic carboxy terminal peptide misc_feature (14) Xaa is any aminoacid 4 Gln Glu Gly Ala Pro Pro Gln Gln Ser Ala Arg Arg Asp Xaa Met Pro 15 10 15 Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 20 25 5 35DNA Oligonucleotide Primer 5 gactggatcc gccatcatgc cattgtcccc cggcc 35 629 DNA Oligonucleotide Primer 6 gactggtacc ggtctgtcat tacacttgc 29 7 25DNA Oligonucleotide Primer 7 gactccatgg ccctgcccct ggagg 25 8 23 DNAOligonucleotide Primer 8 agatcttttg caggaggaga agg 23 9 411 DNA HEBCI67Rmisc_feature (78) n is any nucleotide 9 ggcagaggat gtgcagcagc agggtgggagagaagctcca gtcagcccac aagatgccat 60 tgtcccccgg cctcctgntg ctgctgctctccggggccac ggccaccgct gccctgcccc 120 tggagggtgg ccccaccggc cgagaacagcnagcatatgc aggaagcggc aggaataagg 180 aaaagcagcc tcctgacttt cctcgcttggtggtttgagt ggacctccca ggccagtntc 240 gngcccctca taaggagagg aagcccgggaaggtttgcca ggnggcagga ngggcgcacc 300 cccccagcaa tctgcggggc cgggtacagaattgccntgc aggaaatttn tttctgggaa 360 ganctttttn ctgctggcaa ataaaaacntnaacccatga attnttcaag g 411 10 302 DNA HSVCB08RA misc_feature (199) n isany nucleotide 10 ggcacgagct cgggccagcc aagaacacta caaagttgag ccgtgtgtcctgtccaagct 60 gtgaagtgca acacacgtgc acacacacac accatgcagg ccaaaaagccaacaagagac 120 agagagtggg ctagcgtgtg gccacgtcgg gactgtactc acctcgctgtctcggccggt 180 ggggccaccc tccaggggnc agggcagcgg tggccgtggc cccggnagagcagcagcagc 240 aggnangccg ggggacaatg gncatcttgt gggctgactg gagcttntntgcccaccctg 300 nt 302 11 456 DNA Homo sapiens misc_feature (49) n is anynucleotide 11 tgctgcttaa tacattttat tcaggtctgt cattacactt gcgtgaatntcatcgggtga 60 ggttttattt gcaggaggag aaggtcttcc agaagaagtt cctgcagggcattctgtccc 120 ggcgcgcgga ttgctggggg ggtgcgcctt cctgccgcct tgncacctcccgagcttcct 180 ctcctatgag gggcccggca ctggcctggg gaggtccact caaaccaccaagcgaggaaa 240 gtcaggaggc tgcttttcct tattcctgcc gcttcctgca tatgctcgctgtctcggccg 300 gtggggcacc ctccaggggc agggcagcgg tggccgtggc ccggaagagcagcagcagca 360 ggaggccggg ggacaatggc atcttgtggg ctgactggaa cttctctcccancctgctgc 420 tgctcttcct tcctggcagc cctgaaatca atgttt 456 12 424 DNAHuman Cortistatin CDS (46)..(360) sig_peptide (46)..(102) mat_peptide(103)..(360) misc_feature (71) Xaa is arginine or lysine 12 aagagcagcagcagggtggg agagaagctc cagtcagccc acaag atg cca ttg tcc 57 Met Pro LeuSer ccc ggc ctc ctg ctg ctg ctg ctc tcc ggg gcc acg gcc acc gct gcc 105Pro Gly Leu Leu Leu Leu Leu Leu Ser Gly Ala Thr Ala Thr Ala Ala -15 -10-5 -1 1 ctg ccc ctg gag ggt ggc ccc acc ggc cga gac agc gag cat atg cag153 Leu Pro Leu Glu Gly Gly Pro Thr Gly Arg Asp Ser Glu His Met Gln 5 1015 gaa gcg gca gga ata agg aaa agc agc ctc ctg act ttc ctc gct tgg 201Glu Ala Ala Gly Ile Arg Lys Ser Ser Leu Leu Thr Phe Leu Ala Trp 20 25 30tgg ttt gag tgg acc tcc cag gcc agt gcc ggg ccc ctc ata gga gag 249 TrpPhe Glu Trp Thr Ser Gln Ala Ser Ala Gly Pro Leu Ile Gly Glu 35 40 45 gaagcc cgg gag gtg gcc agg cgg cag gaa ggc gca ccc ccc cag caa 297 Glu AlaArg Glu Val Ala Arg Arg Gln Glu Gly Ala Pro Pro Gln Gln 50 55 60 65 tctgcg cgc cgg gac ara atg ccc tgc agg aac ttc ttc tgg aar acc 345 Ser AlaArg Arg Asp Xaa Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 70 75 80 tty tcctcc tgc aaa taaaacctca cccatgaatg ctcacgcaag tgtaatgaca 400 Phe Ser SerCys Lys 85 gacctgaata aaatgtatta agca 424 13 105 PRT Human Cortistatinmisc_feature (71) Xaa is arginine or lysine 13 Met Pro Leu Ser Pro GlyLeu Leu Leu Leu Leu Leu Ser Gly Ala Thr -15 -10 -5 Ala Thr Ala Ala LeuPro Leu Glu Gly Gly Pro Thr Gly Arg Asp Ser -1 1 5 10 Glu His Met GlnGlu Ala Ala Gly Ile Arg Lys Ser Ser Leu Leu Thr 15 20 25 Phe Leu Ala TrpTrp Phe Glu Trp Thr Ser Gln Ala Ser Ala Gly Pro 30 35 40 45 Leu Ile GlyGlu Glu Ala Arg Glu Val Ala Arg Arg Gln Glu Gly Ala 50 55 60 Pro Pro GlnGln Ser Ala Arg Arg Asp Xaa Met Pro Cys Arg Asn Phe 65 70 75 Phe Trp LysThr Phe Ser Ser Cys Lys 80 85 14 426 DNA Human Cortistatin 14 aagagcagcagcagggtggg agagaagctc cagtcagccc acaagatgcc attgtccccc 60 ggcctcctgctgctgctgct ctccggggcc acggccaccg ctgccctgcc cctggagggt 120 ggccccaccggccgagacag cgagcatatg caggaagcgg caggaataag gaaaagcagc 180 ctcctgactttcctcgcttg gtggtttgag tggacctccc aggccagtgc cgggcccctc 240 ataggagaggaagcccggga ggtggccagg cggcaggaag gcgcaccccc ccagcaatct 300 gcgcgccgggacaraatgcc ctgcaggaac ttcttctgga araccttytc ctcctgcaaa 360 taaaacctcacccatgaatg ctcacgcaag tgtaatgaca gacctgaata aaatgtatta 420 agcagc 426

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide comprising amino acids from about −19 toabout 86 in SEQ ID NO:2; (b) a nucleotide sequence encoding apolypeptide comprising amino acids from about −18 to about 86 in SEQ IDNO:2; (c) a nucleotide sequence encoding a polypeptide comprising aminoacids from about 1 to about 86 in SEQ ID NO:2; (d) a nucleotide sequenceencoding a polypeptide comprising amino acids from about 58 to about 86in SEQ ID NO:2; (e) a nucleotide sequence encoding a polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97639; (f) a nucleotide sequence encoding the mature HumanCortistatin polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97639; and (g) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e), or (f); wherein the isolated nucleic acid moleculedoesn't have a sequence selected from SEQ ID NO:9 or SEQ ID NO:11.
 2. Anisolated nucleic acid molecule comprising a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence identical to a nucleotide sequence in (a),(b), (c), (d), (e), (f), or (g) of claim 1 wherein said polynucleotidewhich hybridizes does not hybridize under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence consistingof only A residues or of only T residues.
 3. A method for making arecombinant vector comprising inserting an isolated nucleic acidmolecule of claim 1 into a vector.
 4. A recombinant vector produced bythe method of claim
 3. 5. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 4 into a hostcell.
 6. A recombinant host cell produced by the method of claim
 5. 7. Arecombinant method for producing a Human Cortistatin polypeptide,comprising culturing the recombinant host cell of claim 6 underconditions such that said polypeptide is expressed and recovering saidpolypeptide.
 8. An isolated Human Cortistatin polypeptide having anamino acid sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) amino acids from about −19 to about 86 inSEQ ID NO:2; (b) amino acids from about −18 to about 86 in SEQ ID NO:2;(c) amino acids from about 1 to about 86 in SEQ ID NO:2; (d) amino acidsfrom about 58 to about 86 in SEQ ID NO:2; (e) the amino acid sequence ofthe Human Cortistatin polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 97639; (f) the aminoacid sequence of the mature Human Cortistatin polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97639; and (g) the amino acid sequence of an epitope-bearing portionof any one of the polypeptides of (a), (b), (c), (d), (e), or (f).
 9. Anisolated antibody that binds specifically to a Human Cortistatinpolypeptide of claim
 8. 10. A method for treatment of an individual inneed of an increased level of Cortistatin activity comprisingadministering to said individual a composition comprising an isolatedpolypeptide of claim
 8. 11. A method useful during the diagnosis of asleep disorder in an individual comprising: (a) measuring Cortistatingene expression level in cells or body fluid of said individual; (b)comparing the Cortistatin gene expression level of said individual witha standard Human Cortistatin gene expression level, whereby an increaseor decrease in the Human Cortistatin gene expression level of saidindividual compared to said standard expression level is indicative of asleep disorder.
 12. An isolated nucleic acid molecule comprising apolynucleotide having a sequence at least 95% identical to a sequenceselected from the group consisting of: (a) the nucleotide sequence of afragment of the sequence shown in SEQ ID NO:1, wherein said fragmentcomprises at least 20 contiguous nucleotides of SEQ ID NO:1, providedthat said nucleotide sequence is not SEQ ID NO:9, SEQ ID NO:10, or SEQID NO:11 or any subfragment thereof; and (b) a nucleotide sequencecomplementary to a nucleotide sequence in (a).
 13. The nucleotidesequence of a fragment of claim 12, wherein said fragment comprises atleast 50 contiguous nucleotides of SEQ ID NO:1.